Both hypercholesterolemia and aging are related to cognitive decline or Alzheimer’s disease. However, their interactive influence on the neurodegenerative progress remains unclear. To address this issue, 6-month-old and 16-month-old female mice were fed a 3% cholesterol diet for 8 weeks, followed by hippocampus-related functional, pathological, biochemical and molecular analyses. The high cholesterol diet did not exacerbate age-dependent cognitive decline and hippocampal neuronal death, and even greatly mitigated decreases of synaptophysin and growth associated protein 43 expression in the hippocampus of aged mice. Compared with young controls, aged mice fed normal diet showed mild activation of hippocampal microglia with increased expression of CD68, a marker of the microglial M1 phenotype, and decreased expression of CD206, a marker of the microglial M2 phenotype. More interestingly, the high cholesterol diet not only improved NLRP3 inflammasome activation and IL-1β expression, but also increased levels of anti-inflammatory cytokines IL-4 and IL-6 in the hippocampus of old mice, suggesting playing pro- and anti-neuroinflammatory effects. In addition, the cholesterol rich diet resulted in a defect of the blood-brain barrier of aged hippocampus, as revealed by increased brain albumin content. These results have revealed both harmful and protective effects of high cholesterol diet on aged brain, which helps us to understand that hypercholesterolemia in the aged population is not associated with dementia and cognitive impairment.
Figure 1. Serum cholesterol analysis of young and aged female mice fed a normal or high-cholesterol diet for 8 weeks. (A) Total cholesterol (TC). (B) High-density lipoprotein cholesterol (HDL-C). (C) Low-density lipoprotein cholesterol (LDL-C). Results were expressed as mean ± SEM. Tests were performed in duplicate on 8 serum samples per group. *P < 0.05, **P < 0.01, ***P < 0.001, high cholesterol diet (HC) mice versus normal control (ctrl) diet mice; #P < 0.05, ##P < 0.01, ###P < 0.001, aged mice versus young mice.
Figure 2. Cognitive analysis of young and aged female mice fed a normal or high-cholesterol diet for 8 weeks. (A) The percentage of time spent in the novel arm of the Y-maze. (B) The numbers of entries into the novel arm of the Y-maze. (C) The mean escape latency during the hidden platform training period of the Morris water maze test. (D) Swimming speed. (E) The percentage of time spent in the target quadrant. (F) The number of crossing the platform area. Results were expressed as mean ± SEM from 8 mice per group. #P < 0.05, aged mice versus young mice.
Figure 3. Pathological analysis of hippocampal neurons of young and aged female mice fed a normal or high-cholesterol diet for 8 weeks. (A) Nissl staining. Degenerative (dark) neurons were densely stained (arrowheads). (B) The percentage of dark neurons. (C) Immunostaining for cleaved-caspase 3. A few apoptotic neurons were marked by arrowheads. (D) The percentage of cleaved-caspase 3 positive neurons. Results were expressed as mean ± SEM of tests in duplicate on 4 hippocampal samples per group. ##P < 0.01, aged mice versus young mice.
Figure 4. Analyses of synapse-related protein levels in the hippocampus of young and aged female mice fed a normal or high-cholesterol diet for 8 weeks. (A) Representative immunoblot bands of SYP, GAP43, PSD-95 and CAMKII. (B) The corresponding densitometry analysis. Results were expressed as mean ± SEM of tests in duplicate on 4 hippocampal samples per group. *P < 0.05, **P < 0.01, high cholesterol diet (HC) mice versus normal control (ctrl) diet mice; #P < 0.05, ## P < 0.01. ### P < 0.001, aged mice versus young mice.
Figure 5. Analyses of glial activation and levels of pro-inflammatory and anti-inflammatory factors in the hippocampus of young and aged female mice fed a normal or high-cholesterol diet for 8 weeks. (A) Immunohistochemical staining for GFAP. (B) Immunohistochemical staining for Iba-1. (C) Cellular surface of GFAP positive astrocytes and Iba-1 positive microglia. (D) Cell counts of GFAP positive astrocytes and Iba-1 positive microglia. (E) The quantitative real-time PCR analysis of mRNA levels of IL-1β, IL-6 and TNF-α. (F) The quantitative real-time PCR analysis of mRNA levels of IL-4 and IL-10. Results were expressed as mean ± SEM of tests in duplicate on 4 hippocampal samples per group. *P < 0.05, ***P < 0.001, high cholesterol diet (HC) mice versus normal control (ctrl) diet mice; #P < 0.05, ###P < 0.001, aged mice versus young mice.
Figure 6. Levels of CD68 and CD206 in the hippocampus of young and aged female mice fed a normal or high-cholesterol diet for 8 weeks. (A) Representative immunoblot for CD68 and CD206. (B) The corresponding densitometry analysis. Results were expressed as mean ± SEM of tests in duplicate on 4 hippocampal samples per group. *P < 0.05, high cholesterol diet (HC) mice versus normal control (ctrl) diet mice; #P < 0.05, aged mice versus young mice.
Figure 7. Activation of NLRP3 inflammasomes in the hippocampus of young and aged female mice fed a normal or high-cholesterol diet for 8 weeks. (A and B) Representative immunoblot of proteins involved in the first and second signal pathways of NLRP3 inflammasome activation. (C and D) The corresponding densitometry analysis. Results were expressed as mean ± SEM of tests in duplicate on 4 hippocampal samples per group. *P < 0.05, **P < 0.01, high cholesterol diet (HC) mice versus normal control (ctrl) diet mice; #P < 0.05, ##P < 0.01, ###P < 0.001, aged mice versus young mice.
Figure 8. Analyses of BBB related protein levels in the hippocampus of young and aged female mice fed a normal or high-cholesterol diet for 8 weeks. (A) Representative immunoblot bands of ZO-1, serum albumin and CD31. (B) The corresponding densitometry analysis. Results were expressed as mean ± SEM of tests in duplicate on 4 hippocampal samples per group. *P < 0.05, high cholesterol diet (HC) mice versus normal control (ctrl) diet mice; #P < 0.05, ###P < 0.01, aged mice versus young mice.
Grouleff J, Irudayam SJ, Skeby KK, Schiøtt B (2015). The influence of cholesterol on membrane protein structure, function, and dynamics studied by molecular dynamics simulations. Biochim Biophys Acta, 1848(9):1783-1795.
Ma X, Feng Y (2016). Hypercholesterolemia tunes hematopoietic stem/progenitor cells for inflammation and atherosclerosis. Int J Mol Sci, 17(7): pii: E1162.
Aguilar D, Fernandez ML (2014). Hypercholesterolemia induces adipose dysfunction in conditions of obesity and nonobesity. Adv Nutr, 5(5): 497-502.
van Rooy MJ, Obesity Pretorius E. (2014). Hypertension and hypercholesterolemia as risk factors for atherosclerosis leading to ischemic events. Curr Med Chem, 21(19): 2121-2129.
Liu JP, Tang Y, Zhou S, Toh BH, McLean C, Li H (2010). Cholestrol involvement in the pathogenesis of neurodegenerative diseases. Mol Cell Neurosci, 43(1): 33-42.
Humpel C (2011). Chronic mild cerebrovascular dysfunction as a cause for Alzheimer’s disease? Exp Gerontol, 46(4): 225-232.
Stozická Z, Zilka N, Novák M (2007). Risk and protective factors for sporadic Alzheimer’s disease. Acta Virol, 51(4): 205-222.
Whitmer RA, Sidney S, Selby J, Johnston SC, Yaffe K (2005). Midlife cardiovascular risk factors and risk of dementia in late life. Neurology, 64(2): 277-281.
Cartocci V, Servadio M, Trezza V, Pallottini V (2016). Can cholesterol metabolism affect brain function and behavior? J Cell Physiol. 232(2): 281-286.
Schreurs BG (2010). The effects of cholesterol on learning and memory. Neurosci Biobehav Rev, 34(8): 1366-1379.
Dietschy JM, Turley SD (2001). Cholesterol metabolism in the brain. Curr Opin Lipidol, 12(2): 105-112.
Gamba P, Testa G, Gargiulo S, Staurenghi E, Poli G, Leonarduzzi G (2015). Oxidized cholesterol as the driving force behind the development of Alzheimer’s disease. Front Aging Neurosci 7:119.
Testa G, Staurenghi E, Zerbinati C, Gargiulo S, Iuliano L, Giaccone G, et al. (2016). Changes in brain oxysterols at different stages of Alzheimer’s disease: Their involvement in neuroinflammation. Redox Biol, 10:24-33.
Martin MG, Ahmed T, Korovaichuk A, Venero C, Menchón SA, Salas I, et al. (2014). Constitutive hippocampal cholesterol loss underlies poor cognition in old rodents. EMBO Mol Med, 6(7):902-917.
Pérez-Cañamás A, Sarroca S, Melero-Jerez C, Porquet D, Sansa J, Knafo S, et al. (2016). A diet enriched with plant sterols prevents the memory impairment induced by a cholesterol loss in senescence-accelerated mice. Neurobiol Aging, 48:1-12.
Reitz C, Luchsinger J, Tang MX, Manly J, Mayeux R (2005). Impact of plasma lipids and time on memory performance in healthy elderly without dementia. Neurology, 64(8): 1378-1383.
van den Kommer TN, Dik MG, Comijs HC, Fassbender K, Lütjohann D, Jonker C (2009). Total cholesterol and oxysterols: early markers for cognitive decline in elderly? Neurobiol Aging, 30(4): 534-545.
Chen YL, Wang LM, Chen Y, Gao JY, Marshall C, Cai ZY, et al. (2016). Changes in astrocyte functional markers and β-amyloid metabolism-related proteins in the early stages of hypercholesterolemia. Neuroscience, 316: 178-191.
Wang Q, Xu Z, Tang J, Sun J, Gao J, Wu T, et al. (2013). Voluntary exercise counteracts Ab25-35-induced memory impairment in mice. Behav Brain Res, 256:618-625.
Xu Z, Xiao N, Chen Y, Huang H, Marshall C, Gao J, et al. (2015). Deletion of aquaporin-4 in APP/PS1 mice exacerbates brain Aβ accumulation and memory deficits. Mol Neurodegener, 10: 58.
Li L, Xiao N, Yang X, Gao J, Ding J, Wang T, et al. (2012). A high cholesterol diet ameliorates hippocampus-related cognitive and pathological deficits in ovariectomized mice. Behav Brain Res, 230(1): 251-258.
Calvo-Ochoa E, Hernández-Ortega K, Ferrera P, Morimoto S, Arias C (2014). Short-term high-fat-and-fructose feeding produces insulin signaling alterations accompanied by neurite and synaptic reduction and astroglial activation in the rat hippocampus. Hippocampus, 34(6): 1001-1008.
Jansen D, Janssen CI, Vanmierlo T, Dederen PJ, van Rooij D, Zinnhardt B, et al. (2012). Cholesterol and synaptic compensatory mechanisms in Alzheimer’s disease mice brain during aging. J Alzheimers Dis, 31(4): 813-826.
Fukata M, Sekiya A, Murakami T, Yokoi N, Fukata Y (2015). Postsynaptic nanodomains generated by local palmitoylation cycles. Biochem Soc Trans, 43(2): 199-204.
Freeman LR, Small BJ, Bickford PC, Umphlet C, Granholm AC (2011). A high-fat/high-cholesterol diet inhibits growth of fetal hippocampal transplants via increased inflammation. Cell Transplant, 20(10):1499-1514.
Thirumangalakudi L, Prakasam A, Zhang R, Bimonte-Nelson H, Sambamurti K, Kindy MS, et al. (2008). High cholesterol-induced neuroinflammation and amyloid precursor protein processing correlate with loss of working memory in mice. J Neurochem, 106(1): 475-485.
Nakagawa Y, Chiba K (2015). Diversity and plasticity of microglial cells in psychiatric and neurological disorders. Pharmacol Ther, 154:21-35.
Pierre WC, Smith PL, Londono I, Chemtob S, Mallard C, Lodygensky GA (2017). Neonatal microglia: The cornerstone of brain fate. Brain Behav Immun, 59:333-345.
Patel AR, Ritzel R, McCullough LD, Liu F (2013). Microglia and ischemic stroke: a double-edged sword. Int J Physiol Pathophysiol Pharmacol, 5(2):73-90.
Varnum MM, Ikezu T (2012). The classification of microglial activation phenotypes on neurodegeneration and regeneration in Alzheimer’s disease brain. Arch Immunol Ther Exp (Warsz), 60(4): 251-266.
Walsh JG, Muruve DA, Power C (2014). Inflammasomes in the CNS. Nat Rev Neurosci, 15(2): 84-97.
Schroder K, Tschopp J (2010). The inflammasomes. Cell, 140(6): 821-832.
Dias IH, Polidori MC, Griffiths HR (2014). Hypercholesterolaemia-induced oxidative stress at the blood-brain barrier. Biochem Soc Trans, 42(4): 1001-1005.
Goodall EF, Wang C, Simpson JE, Baker DJ, Drew DR, Heath PR, et al. (2017). Age-associated changes in the blood brain barrier: Comparative studies in human and mouse. Neuropathol Appl Neurobiol, doi: 10.1111/nan.12408.
Dietschy JM, Turley SD (1993). Serum proteins bypass the blood-brain fluid barriers for extracellular entry to the central nervous system. Exp Neurol, 120(2): 245-263.
Balbuena P, Li W, Ehrich M (2011). Assessments of tight junction proteins occludin, claudin 5 and scaffold proteins ZO1 and ZO2 in endothelial cells of the rat blood-brain barrier: cellular responses to neurotoxicants malathion and lead acetate. Neurotoxicology, 32(1): 58-67.
Almutairi MM, Gong C, Xu YG, Chang Y, Shi H (2016). Factors controlling permeability of the blood-brain barrier. Cell Mol Life Sci, 73(1):57-77
Solomon A, Kivipelto M, Wolozin B, Zhou J, Whitmer RA (2009). Mildlife serum cholesterol and increased risk of Alzheimer’s and vascular dementia three decades later. Dement Geriatr Cogn Disord, 28(1):75-80.
Reitz C, Tang MX, Luchsinger J, Mayeux R (2004). Relation of plasma lipids to Alzheimer disease and vascular dementia. Arch Neurol, 61(5):705-714.
Mielke MM, Zandi PP, Sjögren M, Gustafson D, Ostling S, Steen B (2005). High total cholesterol levels in late life associated with a reduced risk of dementia. Neurology, 64(10):1689-1695.
Corley J, Starr JM, Deary IJ (2015). Serum cholesterol and cognitive functions: the Lothian Birth Cohort 1936. Int Psychogeriatr, 27(3): 439-453.
Bates KA, Sohrabi HR, Rainey-Smith SR, Weinborn M, Bucks RS, Rodrigues M, et al. (2017). Serum highdensity lipoprotein is associated with better cognitive function in a cross-sectional study of aging women. Int J Neurosci, 127(3): 243-252.
Kontush A. (2014). HDL-mediated mechanisms of protection in cardiovascular disease. Cardiovasc Res, 103(3): 341-349.
Murman DL (2015). The impact of age on cognition. Semin Hear, 36(3): 111-121.
Lu J, Wu DM, Zheng ZH, Zheng YL, Hu B, Zhang ZF (2011). Troxerutin protects against high cholesterol-induced cognitive deficits in mice. Brain, 134(Pt3):783-797.
Morrison JH, Baxter MG (2011). The ageing cortical synapse: hallmarks and implication for cognitive decline. Nature Rev Neurosci, 13(4): 240-250.
Villeda SA, Plambeck KE, Middeldorp J, Castellano JM, Mosher KI, Luo J, et al (2014). Young blood reverses age-related impairment in cognitive function and synaptic plasticity in mice. Nature Med, 20(6): 659-663.
Irie M, Hata Y, Takeuchi M, Ichtchenko K, Toyoda A, Hirao K, et al. (1997). Binding of Neuroligins to PSD-95. Science, 277(5331),1511-1515.
Wu DM, Lu J, Zheng YL, Zhou Z, Shan Q, Ma DF (2008). Purple sweet potato color repairs d-galactose-induced spatial learning and memory impairment by regulating the expression of synaptic proteins. Neurobiol Learn Mem, 90(1):19-27.
Koudinov AR, Koudinova NV (2001). Essential role for cholesterol in synaptic plasticity and neuronal degeneration. FASEB J, 15(10):1858-1860.
Poirier J, Baccichet A, Dea D, Gauthier S (1993). Cholesterol synthesis and lipoprotein reuptake during synaptic remodeling in hippocampus in adult rat. Neuroscience, 55(1): 81-90.
Ries M, Sastre M (2016). Mechanisms of Aβ clearance and degradation by glial cells. Front Aging Neurosci, 8: 160.
Vance JE, Hayashi H, Karten B (2005). Cholesterol homeostasis in neurons and glia cells. Semin Cell Dev Biol, 16(2):193-212.
Clarke LE, Barres BA (2013). Emerging roles of astrocytes in neural circuit development. Nat Rev Neurosci, 14(6): 311-321.
Waisman A, Ginhoux F, Greter M, Bruttger J (2015). Homeostasis of microglia in the adult brian: Review of novel microglia depletion systems. Trends Immunol, 36(10):625-636.
Graeber MB, Li W, Rodriguez ML (2011). Role of microglia in CNS inflammation. FEBS Lett, 585(23): 3798-3805.
Wu Z, Yu J, Zhu A, Nakanishi H (2016). Nutrients, microglia aging, and brain aging. Oxid Med Cell Longev, 2016:7498528.
Saeed AA, Genové G, Li T, Lütjohann D, Olin M, Mast N, et al. (2014). Effects of a disrupted blood-brain barrier on cholesterol homeostasis in the brain. J Biol Chem, 289(34): 23712-23722.
Peri A, Benvenuti S, Luciani P, Deledda C, Cellai I (2011). Membrane cholesterol as a mediator of the neuroprotective effects of estrogens. Neuroscience, 191: 107-117.
Peri A, Danza G, Benvenuti S, Luciani P, Deledda C, Rosati F, et al. (2009). New insights on the neuroprotective role of sterols and sex steroids: the seladin-1/DHCR24 paradigm. Front Neuroendocrinol, 30(2): 119-129
Henderson VW (2000). Oestrogens and dementia. Novartis Found Symp, 230:254-265.
Jaya Prasanthi RP, Schommer E, Thomasson S, Thompson A, Feist G, Ghribi O (2008). Regulation of beta-amyloid levels in the brain of cholesterol-fed rabbit, a model system for sporadic Alzheimer’s disease. Mech Ageing Dev, 129(11):649-655.
Ghribi O, Larsen B, Schrag M, Herman MM (2006). High cholesterol content in neurons increases BACE, beta-amyloid, and phosphorylated tau levels in rabbit hippocampus. Exp Neurol, 200(2):460-467.
Kim J, Yoon H, Chung DE, Brown JL, Belmonte KC, Kim J (2016). miR-186 is decreased in aged brain and suppresses BACE1 expression. J Neurochem, 137(3): 436-445.
Mukda S, Panmanee J, Boontem P, Govitrapong P (2016). Melatonin administration reverses the alteration of amyloid precursor protein-cleaving secretases expression in aged mouse hippocampus. Neurosci Lett, 621:39-46.
Nishimura M, Nakamura S, Kimura N, Liu L, Suzuki T, Tooyama I (2012). Age-related modulation of γ-secretase activity in non-human primate brains. J Neurochem 123(1): 21-28.
Eckman EA, Eckman CB (2005). Abeta-degrading enzymes: modulators of Alzheimer’s disease pathogenesis and targets for therapeutic intervention. Biochem Soc Trans, 33(Pt 5):1101-1105.